Phospholipase A2 (PLA2) constitutes a super-family of enzymes that catalyze the hydrolysis of the fatty acid ester from the sn-2 position of membrane phospholipids, yielding a free fatty acid and a lysophospholipid. Among the intracellular PLA2s are the cytosolic Group IVA PLA2 (GIVA PLA2, also referred to herein as cPLA2), which is generally considered a pro-inflammatory enzyme; the calcium-independent Group VIA PLA2 (GVIA PLA2, also referred to herein as iPLA2); and, secreted Group V PLA2 (sPLA2). GVIA PLA2 is actually a group of cytosolic enzymes ranging from 85 to 88 kDa and expressed as several distinct splice variants of the same gene, only two of which have been shown to be catalytically active (Group VIA-1 and VIA-2 PLA2); (Larsson, et al., J. Biol. Chem. 273: 207-214, 1998). The role of GVIA PLA2 in the inflammatory process is unclear, but this enzyme appears to be the primary PLA2 for basal metabolic functions within the cell, reportedly including membrane homeostasis (Balsinde, et al., Proc. Natl. Acad. Sci. U.S.A., 92:8527-8531, 1995; Balsinde, et al., J. Biol. Chem., 272: 29317-29321, 1997; Balsinde, et al., J. Biol. Chem., 272:16069-16072, 1997; Ramanadham, et al., J. Biol. Chem., 274:13915-13927, 1999; Birbes, et al., Eur. J. Biochem., 267:7118-7127, 2000; and Ma, et al., Lipids, 36:689-700, 2001), insulin receptor signaling (Ramanadham, et al., J. Biol. Chem., 274: 13915-13927, 1999; Ma, et al., J. Biol. Chem., 276: 13198-13208, 2001) and calcium channel regulation. (Guo, et al., J. Biol. Chem., 277: 32807-32814, 2002; Cummings, et al., Am. J. Physiol. Renal Physiol., 283: F492-498, 2002). GVIA, GIVA and GV PLA2 are all present and play active roles in central nervous system inflammatory processes (see, e.g., Sun, et al., J. Lipid Res., 45: 205-213, 2004).
The GVIA PLA2 enzymes all contain a consensus lipase motif, Gly-Thr-Ser*-Thr-Gly, with the catalytic serine confirmed by site-directed mutagenesis (Larsson, et al., J. Biol. Chem., 273:207-14, 1998; Tang, et al., J. Biol. Chem., 272: 8567-8575, 2002). Other residues critical for catalysis have yet to be confirmed, and while the mechanism by which it cleaves the sn-2 linkage has not been established, GVIA PLA2 is likely to be an hydrolase with a catalytic Ser/Asp dyad similar to Group IVA PLA2 (Dessen, et al., Cell 1999, 97: 349-360, 1999; Dessen, Biochim. Biophys. Acta, 1488:40-47, 2000; Phillips, et al., J. Biol. Chem., 278: 41326-41332, 2003). Constitutive mRNA and protein have been detected in the spinal cord for group IVA calcium-dependent PLA2 (Group IVA cPLA2) and Group VIA calcium-independent iPLA2 (Group VIA iPLA2) and secretory Group II and V sPLA2 forms (Lucas, et al., Br. J. Pharmacol., 144:940-952, 2005, Svensson et al., Annu. Rev. Pharmacol. Toxicol., 42:553-555, 2005).
The discovery of a novel structural series of 2-oxoamides that inhibit Group IVA cPLA2 in vitro and in vivo (Kokotos, et al., J. Med. Chem., 45:2891-2893, 2002; Kokotos, et al., J. Med. Chem., 47:3615-3628, 2004) was recently reported. In that initial work, 2-oxoamides were observed to inhibit inflammation in the rat paw carrageenan-induced edema assay (Kokotos, et al., supra, 2004).
Based upon the similarity of substrates, classes of common inhibitors, very limited sequence homology in the region of the catalytic serine, and similarities in the active sites of GIVA and GVIA PLA2, GIVA PLA2 may show cross-reactivity with GVIA PLA2. It has been difficult, therefore, to design GIVA and GVIA PLA2 selective inhibitors that can distinguish between the molecules in vivo. Further, selective inhibitors for GV PLA2 have been difficult to design. However, as the results reported herein demonstrate, inhibition to PLA2 molecules can have surprisingly different consequences in vivo. PLA2 inhibitors that are selective for particular targets can be used to advantage to treat disease processes related to PLA2 metabolism. Here, the use of compounds specific for particular PLA2 s is demonstrated in treating multiple sclerosis and spinal cord injury.